ARE-mediated Gene Expression Research Articles

MOLECULAR MECHANISMS OF β-AMYLOID-INDUCED INHIBITION OF GLUTATHIONE SYNTHESIS IN RATS

Oxidative stress is one of the main pathogenic mechanisms of Alzheimer's disease (AD). Glutathione (GSH) is an endogenous antioxidant protecting cells from oxidative injury. However, it is reported that the content of GSH is lower both in the brain of AD patients and the AD animal models. The reason why GSH content is lower in such an oxidative environment of AD brain is not clear. Glutamatecysteine ligase (GCL), composed of a catalytic (GCLC) and a modifier (GCLM) subunit, is the rate-limiting enzyme in GSH synthesis. The transcription of both GCLC and GCLM are regulated by Keap1-Nrf2-ARE signal pathway. Meanwhile, as a chaperone protein of Nrf2, small Maf proteins form heterodimers with Nrf2 leading to activation or repression of ARE-mediated gene expression. The current study aimed at investigating the molecular mechanisms of regulation on GCL transcription by amyloid-β (Aβ) involving Keap1-Nrf2-ARE signal pathway. Aβ intrahippocampal injection rat model and human neuroblastoma SH-SY5Y cells treated with Aβ were used. The expressions of Nrf2, GCLC, GCLM and small Maf proteins as well as GSH level under Aβ insult at different time point were tested. Overexpression and knockdown of Maf proteins were used to explore the roles of Maf proteins on GCL expression. The banding of Keap1 and Nrf2 was examined by CoIP. The expressions of GCLC, GCLM and GSH level were increased in acute stage of Aβ treatment (within 6h), while the expressions of GCLC, GCLM and GSH level were decreased accompanying with activation of Keap1-Nrf2-ARE signal pathway after Aβ treatment for 6–24h. The nuclear level of Nrf2 restored to normal level accompanying with down regulation of GCL and low GSH in the late stage of Aβ treatment. The nuclear expressions of MafG and MafK were largely induced and displacement of Nrf2 nuclear binding to ARE in the late stage of Aβ treatment, while, MafG or MafK knockdown restored GCLC expression and GSH level. Meanwhile, the banding of Keap1 and Nrf2 were enhanced with decreased expression of p-Nrf2 Ser40. Aβ-mediated inhibition of Keap1-Nrf2-ARE signal pathway and induction of nuclear Maf proteins contributed to lowering GSH content due to inhibition of GCL expression.

Redox Homeostasis Plays Important Roles in the Maintenance of the Drosophila Testis Germline Stem Cells.

SummaryOxidative stress influences stem cell behavior by promoting the differentiation, proliferation, or apoptosis of stem cells. Thus, characterizing the effects of reactive oxygen species (ROS) on stem cell behavior provides insights into the significance of redox homeostasis in stem cell-associated diseases and efficient stem cell expansion for cellular therapies. We utilized the Drosophila testis as an in vivo model to examine the effects of ROS on germline stem cell (GSC) maintenance. High levels of ROS induced by alteration in Keap1/Nrf2 activity decreased GSC number by promoting precocious GSC differentiation. Notably, high ROS enhanced the transcription of the EGFR ligand spitz and the expression of phospho-Erk1/2, suggesting that high ROS-mediated GSC differentiation is through EGFR signaling. By contrast, testes with low ROS caused by Keap1 inhibition or antioxidant treatment showed an overgrowth of GSC-like cells. These findings suggest that redox homeostasis regulated by Keap1/Nrf2 signaling plays important roles in GSC maintenance.

Abstract 1639: Chemopreventive and anti-inflammatory properties of “supercinnamaldehydes” - novel cinnamyl-based compounds

Abstract Induction of the antioxidant and cytoprotective phase 2 enzymes via the Keap1/Nrf2/antioxidant response element (ARE) pathway is an accepted strategy for cancer prevention. Recent studies have identified inhibition of inflammatory response as a consistent attribute of chemicals with phase 2 enzymes-inducing properties. As chronic inflammation has been associated with the pathogenesis of cancer, compounds eliciting both phase 2 enzymes-inducing and anti-inflammatory properties may serve as potential candidates for cancer chemoprevention. In our previous study, we had found that trans-cinnamaldehyde (CA) and its analog 2-benzoyloxycinnamaldehyde (BCA) were able to induce phase 2 enzymes via the Keap1/Nrf2/ARE pathway. In this study, we investigated the chemopreventive and anti-inflammatory properties of CA, BCA, and a novel series of “supercinnamaldehydes”. In the ARE-luciferase reporter gene assay, compounds were screened for their chemopreventive potencies by determining their ability to induce ARE-mediated gene expression in human embryonic kidney cells (HEK293). All supercinnamaldehydes induced ARE transcriptional activity, with one compound showing an induction of almost nine fold at 20 µM as compared to that of around six fold induction for 40 µM CA. To evaluate the anti-inflammatory properties of supercinnamaldehydes, we induced inflammatory conditions in mouse macrophage-like cells (RAW264.7) with lipopolysachharide (LPS) and measured nitric oxide (NO) concentrations in presence of the compounds. Compounds with good ARE-fold induction also exhibited low IC50 values for NO generation. The most potent compound inhibited NO production with an IC50 of 4.63 µM, significantly lower than the IC50 (34.45 µM) for CA. We focused our subsequent studies on CA, BCA, and two supercinnamaldehydes with highest ARE-fold induction and lowest IC50 values for NO release. The selected compounds showed marked induction of Nrf2 and phase 2 enzyme levels in HEK293 cells and wild-type mouse embryonic fibroblasts (MEFs) as observed by immunoblotting. In contrast, little induction was observed in Keap1-knockout MEFs, suggesting that the compounds could, at least in part, be targeting Keap1/Nrf2/ARE pathway for phase 2 enzyme induction. Immunoblotting also revealed that these compounds inhibited the LPS-induced up-regulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). A suppressive effect on the NFκB signaling pathway was also observed. In conclusion, we hereby introduce a new class of compounds with superior phase 2 enzyme inducing and anti-inflammatory properties than the naturally occurring CA, which may be potentially efficacious in cancer prevention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1639. doi:1538-7445.AM2012-1639

Antioxidant-induced INrf2 (Keap1) tyrosine 85 phosphorylation controls the nuclear export and degradation of the INrf2–Cul3–Rbx1 complex to allow normal Nrf2 activation and repression

INrf2 (Keap1) serves as a negative regulator of the cytoprotective transcription factor Nrf2. At basal levels, INrf2 functions as a substrate adaptor to sequester Nrf2 into the Cul3-Rbx1 E3 ligase complex for ubiquitylation and proteasomal degradation. In response to antioxidants, Nrf2 is released from the INrf2-Cul3-Rbx1 complex and translocates into the nucleus, where it activates ARE-mediated cytoprotective gene expression. The present studies demonstrate that INrf2, Cul3 and Rbx1 export out of the nucleus and are degraded during the early or pre-induction response to antioxidants. Mutation of Tyr85 in INrf2 stymied the nuclear export of INrf2, suggesting that tyrosine phosphorylation controls the pre-induction nuclear export and degradation in response to antioxidants. The nuclear export of Cul3-Rbx1 were also blocked when INrf2Tyr85 was mutated, suggesting that INrf2-Cul3-Rbx1 undergo nuclear export as a complex. INrf2 siRNA also inhibited the nuclear export of Cul3-Rbx1, confirming that Cul3-Rbx1 requires INrf2 for nuclear export. Newly synthesized INrf2-Cul3-Rbx1 is imported back into the nucleus during the post-induction period to ubiquitylate and degrade Nrf2. Mutation of INrf2Tyr85 had no effect on activation of Nrf2 but led to nuclear accumulation of Nrf2 during the post-induction period owing to reduced export and degradation of Nrf2. Our results also showed that nuclear export and degradation followed by the new synthesis of INrf2-Cul3-Rbx1 controls the cellular abundance of the proteins during different phases of antioxidant responses. In conclusion, the early or pre-induction nuclear export of INrf2 in response to antioxidants is controlled by tyrosine phosphorylation, whereas the nuclear export of Cul3 and Rbx1 is controlled by INrf2, allowing normal activation or repression of Nrf2.

Induction of activation of the antioxidant response element and stabilization of Nrf2 by 3-(3-pyridylmethylidene)-2-indolinone (PMID) confers protection against oxidative stress-induced cell death

The antioxidant response elements (ARE) are a cis-acting enhancer sequence located in regulatory regions of antioxidant and detoxifying genes. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a member of the Cap ‘n’ Collar family of transcription factors that binds to the ARE and regulates the transcription of specific ARE-containing genes. Under oxidative stress, Nrf2/ARE induction is fundamental to defense against reactive oxygen species (ROS) and serves as a key factor in the protection against toxic xenobiotics. 3-(3-Pyridylmethylidene)-2-Indolinone (PMID) is a derivative of 2-indolinone compounds which act as protein kinase inhibitors and show anti-tumor activity. However, the role of PMID in the oxidative stress remains unknown. In the present study, we showed that PMID induced the activation of ARE-mediated transcription, increased the DNA-binding activity of Nrf2 and then up-regulated the expression of antioxidant genes such as HO-1, SOD, and NQO1. The level of Nrf2 protein was increased in cells treated with PMID by a post-transcriptional mechanism. Under CHX treatment, the stability of Nrf2 protein was enhanced by PMID with decreased turnover rate. We showed that PMID reduced the ubiquitination of Nrf2 and disrupted the Cullin3 (Cul3)-Keap1 interaction. Furthermore, cells treated with PMID showed resistance to cytotoxicity by H2O2 and pro-oxidant 6-OHDA. PMID also up-regulated the antioxidant level in BALB/c mice. Taken together, the compound PMID induces the ARE-mediated gene expression through stabilization of Nrf2 protein and activation of Nrf2/ARE pathway and protects against oxidative stress-mediated cell death.

Hepatitis C Virus Impairs the Induction of Cytoprotective Nrf2 Target Genes by Delocalization of Small Maf Proteins

The expression of a variety of cytoprotective genes is regulated by short cis-acting elements in their promoters, called antioxidant response elements (AREs). A central regulator of ARE-mediated gene expression is the NF-E2-related factor 2 (Nrf2). Nrf2/ARE-regulated genes are crucial for the maintenance of cellular integrity. Hepatitis C virus inhibits the induction of ARE-regulated genes, but neither induction nor inhibition of ARE-regulated gene expression affects HCV replication directly. In HCV-replicating cells the core protein triggers the delocalization of sMaf proteins from the nucleus to the replicon complex. Here sMafs bind to NS3. The extranuclear sMaf proteins prevent Nrf2 from entry in the nucleus and thereby inhibit the induction of Nrf2/ARE-regulated genes. This results in the decreased expression of cytoprotective genes. Consistent with this finding, the elimination of ROI is impaired in HCV-replicating cells as demonstrated by elevated protein oxidation or 8-OH-dG formation, reflecting DNA damage. In conclusion, these data identified a novel mechanism of Nrf2 regulation and suggest that the HCV-dependent inhibition of Nrf2/ARE-regulated genes confers to the HCV-associated pathogenesis by elevation of intracellular ROI that affect integrity of the host genome and regenerative processes.

Hepatitis B Virus Induces Expression of Antioxidant Response Element-regulated Genes by Activation of Nrf2

The expression of a variety of cytoprotective genes is regulated by short cis-acting elements in their promoters, called antioxidant response elements (AREs). A central regulator of ARE-mediated gene expression is the NF-E2-related factor 2 (Nrf2). Human hepatitis B virus (HBV) induces a strong activation of Nrf2/ARE-regulated genes in vitro and in vivo. This is triggered by the HBV-regulatory proteins (HBx and LHBs) via c-Raf and MEK. The Nrf2/ARE-mediated induction of cytoprotective genes by HBV results in a better protection of HBV-positive cells against oxidative damage as compared with control cells. Furthermore, there is a significantly increased expression of the Nrf2/ARE-regulated proteasomal subunit PSMB5 in HBV-positive cells that is associated with a decreased level of the immunoproteasome subunit PSMB5i. In accordance with this finding, HBV-positive cells display a higher constitutive proteasome activity and a decreased activity of the immunoproteasome as compared with control cells even after interferon α/γ treatment. The HBV-dependent induction of Nrf2/ARE-regulated genes might ensure survival of the infected cell, shape the immune response to HBV, and thereby promote establishment of the infection.

BTZO-1, a Cardioprotective Agent, Reveals that Macrophage Migration Inhibitory Factor Regulates ARE-Mediated Gene Expression

In a screening program to discover therapeutic drugs for heart diseases, we identified BTZO-1, a 1,3-benzothiazin-4-one derivative, which activated antioxidant response element (ARE)-mediated gene expression and suppressed oxidative stress-induced cardiomyocyte apoptosis invitro. An active BTZO-1 derivative for ARE-activation protected heart tissue during ischemia/reperfusion injury in rats. Macrophage migration inhibitory factor (MIF), which is known to protect cells from oxidative insult, was identified as a specific BTZO-1-binding protein. BTZO-1 binds to MIF with a K(d) of 68.6 nM, and its binding required the intact N-terminal Pro1. MIF, in the presence of BTZO-1, activated the glutathione S-transferase Ya subunit (GST Ya) gene ARE, whereas reduction of cellular MIF protein levels by siRNA suppressed BTZO-1-induced GST Ya expression. These results suggest that BTZO-1 activates the GST Ya gene ARE by interacting with MIF.

Redox-dependent signaling system Nrf2/ARE in inflammation

Transcription factor Nrf2 plays a key role in cell defense against oxidative stress, as well as in counteracting chemical and physical factors that induce apoptosis and carcinogenesis. The mechanism of Nrf2/ARE redox regulation and ARE-mediated gene expression are analyzed in the review. Special attention was paid to anti-inflammatory effects of Nrf2/ARE system, which significantly manifest in Nrf2 knockout animal.

Effect of tBHQ and sulforaphane on Nrf2-ARE signaling pathway of Caco2 cells

To investigate the effect of tBHQ and sulforaphane on the protein expression in Nrf2-ARE signaling pathway of Caco2 cells. Human colorectal carcinoma Caco2 cells were treated with 20 micromol/L tBHQ and 5 micromol/L sulforaphane (SFN) respectively. Real time PCR, Western blotting and immunoflourescence staining (IF) were performed to measure the target gene expression. Nrf2, AKR1C1 and NQO1 protein expressions were increased time-dependently in Caco2 cells after treatment with tBHQ and SFN. Time-course experiments showed that tBHQ and SFN increased the accumulation of Nrf2, and concomitantly increased the protein levels of AKR1C1 and NQO1. Real-time PCR and Western blotting showed that tBHQ and SFN significantly increased the expression of Nrf2 at 8h after the treatment, and AKR1C1 and NQO1 at 16 h. Confocal microscopy technique showed that Nrf2 accumulated in the nucleus at 6-8 h after treatment with tBHQ. After 1 h treatment with tBHQ the nuclear Nrf2 maintained at elevated level for at least 4 h with tBHQ withdrawn. tBHQ and SFN induced nuclear accumulation of Nrf2 and activated Nrf2-dependent regulation of ARE-mediated gene expression in Caco2 cells. In addition, the results provide experimental evidence for choosing the dose and frequency of the inducer in cancer chemoprevention study and in developing inhibitors of Nrf2-ARE signaling pathway.

Antioxidant-induced Phosphorylation of Tyrosine 486 Leads to Rapid Nuclear Export of Bach1 That Allows Nrf2 to Bind to the Antioxidant Response Element and Activate Defensive Gene Expression

Antioxidants cause stabilization and nuclear translocation of NF-E2-related factor 2 (Nrf2), where it binds to the antioxidant response element (ARE) and induces up-regulation of defensive genes that protect cells against oxidative and electrophilic stress. Bach1, the negative regulator of Nrf2, competes with Nrf2 for binding to the ARE in the human NQO1 promoter. In this study, we demonstrate that Bach1 exits the nucleus within 1-2 h upon antioxidant treatment. Genistein, an inhibitor of tyrosine kinases, blocked nuclear export of Bach1. Site-directed mutagenesis and immunoprecipitation assays identified tyrosine 486 that was phosphorylated in response to the antioxidant and was essential for nuclear export of Bach1. Chromatin immunoprecipitation assays revealed a competitive interplay between Bach1 and Nrf2 at 1-2 and 4 h for binding to the human NQO1 ARE. Luciferase and real time PCR assays showed a significant decrease in antioxidant induction of reporter activity and mRNA levels in cells transfected with mutant Bach1 compared with wild type. This decrease was due to the absence of nuclear export of the mutant protein. Bach1 levels inside the nucleus returned to normal at 4 h after antioxidant treatment in the absence but not in the presence of protein synthesis inhibitor cycloheximide. In addition, antioxidant treatment increased the transcription of Bach1 as shown by pulse chase and real time PCR experiments. Taken together, these results indicate that increased synthesis of Bach1 restored its nuclear levels to normal at 4 h. In conclusion, antioxidant-induced tyrosine 486 phosphorylation leads to nuclear exit of Bach1, thus allowing Nrf2 access to the ARE.

Dietary flavonoid (−)epicatechin stimulates phosphatidylinositol 3‐kinase‐dependent anti‐oxidant response element activity and up‐regulates glutathione in cortical astrocytes

Flavonoids are plant-derived polyphenolic compounds with neuroprotective properties. Recent work suggests that, in addition to acting as hydrogen donors, they activate protective signalling pathways. The anti-oxidant response element (ARE) promotes the expression of protective proteins including those required for glutathione synthesis (xCT cystine antiporter, gamma-glutamylcysteine synthetase and glutathione synthase). The use of a luciferase reporter (ARE-luc) assay showed that the dietary flavan-3-ol (-)epicatechin activates this pathway in primary cortical astrocytes but not neurones. We also examined the distribution of NF-E2-related factor-2 (Nrf2), a key transcription factor in ARE-mediated gene expression. We found, using immunocytochemistry, that Nrf2 accumulated in the nuclei of astrocytes following exposure to tert-butylhydroquinone (100 microM) and (-)epicatechin (100 nM). (-)Epicatechin signalling via Nrf2 was inhibited by wortmannin implicating a phosphatidylinositol 3-kinase-dependent pathway. Finally, (-)epicatechin increased glutathione levels in astrocytes consistent with an up-regulation of ARE-mediated gene expression. Together, this suggests that flavonoids may be cytoprotective by increasing anti-oxidant gene expression.

The Antioxidant Response Element and Oxidative Stress Modifiers in Airway Diseases

Redox balance is particularly important in the airways because they are the first points of contact with environmental pollutants such as ozone, particles, and cigarette smoke, as well as pathogens such as bacteria and viruses. However, an imbalance between toxicant-induced reactive oxygen (ROS) and nitrogen (RNS) species and the antioxidant defense system leads to oxidative stress, which has been implicated in the development and/or perpetuation of airway diseases, including malignancy. Various antioxidant enzymes and proteins are critical to maintaining the reducing environment of the cell and preventing the damage to various biomolecules that is elicited by ROS/RNS. Emerging evidence indicates that transcriptional activation of the antioxidant response element (ARE) plays a crucial role in modulating oxidative stress and providing cytoprotection against prooxidant stimuli. This review focuses on the regulation and functional roles of key effectors that bind to the ARE and differentially (up- or down-) regulate gene expression in lung tissue/cell types in response to respiratory toxicants. It also provides a perspective on whether boosting ARE-mediated gene expression with dietary plants and synthetic plant products will offer a better therapeutic strategy for mitigating oxidative stress and respiratory pathogenesis.

Epigallocatechin activates haem oxygenase-1 expression via protein kinase Cδ and Nrf2

The Nrf2/anti-oxidant response element (ARE) pathway plays an important role in regulating cellular anti-oxidants, including haem oxygenase-1 (HO-1). Various kinases have been implicated in the pathways leading to Nrf2 activation. Here, we investigated the effect of epigallocatechin (EGC) on ARE-mediated gene expression in human monocytic cells. EGC time and dose dependently increased HO-1 mRNA and protein expression but had minimal effect on expression of other ARE-regulated genes, including NAD(P)H:quinone oxidoreductase 1, glutathione cysteine ligase and ferritin. siRNA knock down of Nrf2 significantly inhibited EGC-induced HO-1 expression. Furthermore, inhibition of PKC by Ro-31-8220 dose dependently decreased EGC-induced HO-1 mRNA expression, whereas MAP kinase and phosphatidylinositol-3-kinase pathway inhibitors had no significant effect. EGC stimulated phosphorylation of PKCαβ and δ in THP-1 cells. PKCδ inhibition significantly decreased EGC-induced HO-1 mRNA expression, whereas PKCα- and β-specific inhibitors had no significant effect. These results demonstrate for the first time that EGC-induced HO-1 expression occurs via PKCδ and Nrf2.

Phosphorylation and Dephosphorylation of Tyrosine 141 Regulate Stability and Degradation of INrf2: A NOVEL MECHANISM IN NRF2 ACTIVATION

INrf2-Nrf2 proteins are sensors of chemical/radiation stress. Nrf2, in response to stresses, is released from INrf2. Nrf2 is translocated into the nucleus where it binds to the antioxidant response element and coordinately activates the expression of a battery of genes that protect cells against oxidative and electrophilic stress. An autoregulatory loop between INrf2 and Nrf2 regulates their cellular abundance. Nrf2 activates INrf2 gene expression, and INrf2 serves as an adapter for degradation of Nrf2. In this report, we demonstrate that mutation of tyrosine 141 in bric-a-bric, tramtrack, broad complex domain to alanine rendered INrf2 unstable and nonfunctional. INrf2Y141A mutant degraded rapidly as compared with wild type INrf2, although it could dimerize and bind Nrf2. De novo synthesized INrf2 protein was phosphorylated at tyrosine 141. Tyrosine 141-phosphorylated INrf2 was highly stable. Treatment with hydrogen peroxide, which is an oxidizing agent, led to dephosphorylation of INrf2Y141, resulting in rapid degradation of INrf2. This resulted in stabilization of Nrf2 and activation of ARE-mediated gene expression. These results demonstrate that stress-induced dephosphorylation of tyrosine 141 is a novel mechanism in Nrf2 activation and cellular protection.

Induction of antioxidant and detoxification response by oxidants in cardiomyocytes: Evidence from gene expression profiling and activation of Nrf2 transcription factor

Mild or low doses of oxidants are known to prime cells towards resistance against further damage. In cardiomyocytes, we found that pretreatment with 100 μM H2O2 prevents the cells from apoptosis induced by doxorubicin (Dox). Affymetrix microarray analyses of 28,000 genes reveal that H2O2 treated cells reduced expression of genes encoding cytochrome c, mitochondrial complex I, III, IV and V and several contractile proteins. Elevated expression of antioxidant and detoxification genes appears as a dominant feature of the gene expression profile of H2O2 treated cells. Most of the genes in this category contain an Antioxidant Response Element (ARE) in their promoters. Measurements of ARE promoter–reporter gene activity indicate a dose- and time-dependent activation of the ARE by H2O2. Since the Nrf2 transcription factor regulates ARE-mediated gene expression, we overexpressed Nrf2 to test whether activation of Nrf2 is sufficient to induce cytoprotection. High levels of Nrf2 expression were achieved via adenovirus mediated gene delivery. Transduced Nrf2 was present in the nuclei and caused an increase in the expression of NAD(P)H:quinone oxidoreductase 1 (NQO1), a representative downstream target of Nrf2. Unlike H2O2 pretreated cells, the cells expressing high levels of Nrf2 were not resistant to Dox-induced apoptosis. Therefore, the cytoprotective effect of H2O2 pretreatment is not reliant upon Nrf2 activation alone as measured by resistance against Dox-induced apoptosis.

Low and High Dose UVB Regulation of Transcription Factor NF-E2-Related Factor 2

Transcription factor NF-E2-related factor 2 (Nrf2) regulates antioxidant response element (ARE)-mediated expression and coordinated induction of chemoprotective proteins in response to chemical stress. In this report, we investigated Nrf2 response to low and high dose UVB irradiation. Low dose (7.5 J/m(2)) UVB exposure of mouse hepatoma, mouse keratinocyte, and human skin fibroblast cells led to the nuclear accumulation of Nrf2 and up-regulation of ARE-mediated gene expression. On the contrary, and intriguingly, high dose (20 J/m(2)) UVB exposure of cells led to the nuclear exclusion of Nrf2 and down-regulation of chemoprotective gene expression with possible implications in UVB carcinogenesis. We investigated the mechanism by which high dose UVB induced the nuclear exclusion of Nrf2. Prior treatment with nuclear export inhibitor, leptomycin B, abrogated the UVB-induced nuclear exclusion of Nrf2, indicating that the decrease of Nrf2 in the nucleus was due to the nuclear export of Nrf2. High dose UVB increased the phosphorylation of Nrf2Y568 which stimulated the nuclear export of Nrf2. Mutation of Nrf2Y568 to phenylalanine and src kinase inhibitor PP2 abrogated/reduced the UVB-induced phosphorylation of Nrf2Y568 and nuclear exclusion of Nrf2. Transfection with src family member Fyn small interfering RNA resulted in the nuclear accumulation of Nrf2 and an increase in the expression and UVB induction of ARE-mediated gene expression. UVB exposure also induced the nuclear localization of Fyn. These results suggest that high dose UVB induced the activation/nuclear localization of Fyn which led to increased phosphorylation of Nrf2Y568 and enhanced nuclear export of Nrf2. This resulted in nuclear exclusion of Nrf2 and down-regulation of ARE-mediated chemoprotective gene expression.

Butylated hydroxyanisole regulates ARE‐mediated gene expression via Nrf2 coupled with ERK and JNK signaling pathway in HepG2 cells

Many natural and synthetic cancer chemopreventive compounds are potent inducers of phase II detoxifying and antioxidant stress responsive genes. The phase II/antioxidant gene expression plays critical role in chemoprevention of carcinogenesis. The antioxidant responsive element (ARE), located on many phase II/antioxidant genes, binds with the transcription factor Nrf2, and is required for the activation of these phase II/antioxidant gene expression induced by many natural and synthetic cancer chemopreventive compounds. In this study, we investigated the potential roles of extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) in the regulation of butylated hydroxyanisole (BHA)-induced and Nrf2-dependent ARE transcriptional activity and ARE-mediated endogenous heme oxygenase-1 (HO-1) protein expression in HepG2 cells. ARE transcriptional activity and HO-1 protein expression were increased dose dependently after treatment with BHA in HepG2 cells. Dose-response and time-course experiments showed that BHA increased the accumulation of Nrf2, and concomitantly decreased the protein level of Keap1. We next examined the phosphorylation of the MAPKs, and found that BHA significantly increased the phosphorylation levels of ERK1/2 and JNK1/2. Importantly BHA-induced ARE transcriptional activity was attenuated by the inhibition of ERK and JNK signaling using biochemical inhibitors and their dominant-negative mutants. Using confocal microscopy technique, treatment with BHA showed the release of Nrf2 sequestered by Keap1 in the cytosol, and that Nrf2 translocated into the nucleus. Importantly, cDNA transfections of ERK and JNK signaling pathways similarly released Nrf2 from Keap1 cytosolic sequestration and translocating Nrf2 into the nucleus. Taken together, these results strongly suggested that ERK and JNK signaling pathways played important and positive roles in BHA-induced and Nrf2-dependent regulation of ARE-mediated gene expression, as well as the nuclear translocation of Nrf2 in HepG2 cells.

Role of protein kinase C δ in curcumin-induced antioxidant response element-mediated gene expression in human monocytes

The Nrf2/antioxidant response element (ARE) signaling pathway plays a key role in activating cellular antioxidants, including heme oxygenase-1 (HO-1), NADPH quinone oxidoreductase-1 (NQO1), and glutathione. Protein kinase C (PKC) may also regulate these antioxidants, as PKC phosphorylates Nrf2 in vitro. This study examined the role of PKC in ARE-mediated gene regulation in human monocytes by curcumin, a potent inducer of the Nrf2/ARE pathway. Curcumin increased HO-1 and glutamyl cysteine ligase modulator (GCLM) expression and stimulated Nrf2 binding to the ARE. Curcumin also rapidly stimulated PKC phosphorylation and Ro-31-8220, a pan-PKC inhibitor, decreased curcumin-induced GCLM and HO-1 mRNA expression and ARE binding. Rottlerin (a PKC δ inhibitor) and PKC δ antisense oligonucleotides significantly inhibited curcumin-induced GCLM and HO-1 mRNA expression and ARE binding. Furthermore, a p38 MAP kinase inhibitor reduced GCLM and HO-1 expression and rottlerin inhibited curcumin-induced p38 phosphorylation. In summary, curcumin activates ARE-mediated gene expression in human monocytes via PKC δ, upstream of p38 and Nrf2.

Nrf2: A Potential Molecular Target for Cancer Chemoprevention by Natural Compounds

One of the most prominent strategies of cancer chemoprevention might be protecting cells or tissues against various carcinogens and carcinogenic metabolites derived from exogenous or endogenous sources. This protection could be achieved through the induction of phase 2 detoxifying enzymes and antioxidant enzymes such as glutathione S-transferase, NAD(P)H quinone oxidoreductase 1, and heme oxygenase-1, a process that is mediated mainly by the antioxidant response elements (ARE) within the promoter regions of these genes. Nuclear factor-erythroid 2-related factor 2 (Nrf2), a member of the Cap 'n' collar (CNC) family of basic region-leucine zipper transcription factors, plays a key role in ARE-mediated gene expression. Under normal condition, Nrf2 is sequestered in the cytoplasm by an actin-binding protein, Kelch-like ECH associating protein 1 (Keap1), and upon exposure of cells to inducers such as oxidative stress and certain chemopreventive agents, Nrf2 dissociates from Keap1, translocates to the nucleus, binds to AREs, and transactivates phase 2 detoxifying and antioxidant genes. Several upstream signaling pathways including mitogen-activated protein kinases, protein kinase C, phosphatidylinositol 3-kinase, and transmembrane kinase are implicated in the regulation of Nrf2/ARE activity. Furthermore, many natural chemopreventive agents are known to induce Nrf2/ARE-dependent gene expression, also in part by regulating the turnover of the Nrf2 protein itself. This review discusses our current understanding of the Nrf2/ARE pathway as a potential molecular target for cancer chemoprevention, as well as the feasibility of screening natural compounds for activation of this pathway and as potential cancer preventive agents for human use.